Norovirus vaccine

ABSTRACT

A dry powder Norovirus vaccine is provided, which comprises at least two Norovirus antigens representing different genogroups. The vaccine may be produced by formulation with a mixture of different antigens or combination of monovalent powders with each containing one antigen. The formulated vaccine is suitable for mucosal administration and soluble in aqueous solutions for parenteral administration. A method of immunization is also provided, which comprises at least one administration of the vaccine via mucosal and/or parental route. The immunization may have multiple administrations of the vaccine, i.e., one or more immunizations via a mucosal route followed by one or more immunizations via a parenteral route or vice versa, to maximize both mucosal and systemic immune responses and protection against Norovirus infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 16/877,015,filed May 18, 2020, which is a divisional of U.S. application Ser. No.15/754,807, filed Feb. 23, 2018, now abandoned, which is the NationalStage of International Application PCT/US2016/048940, filed Aug. 26,2016, which claims priority to U.S. Provisional Patent Application62/211,289, filed Aug. 28, 2015, all of which are hereby incorporated byreference herein.

FIELD OF INVENTION

The present invention is generally related to vaccines for prevention ofinfectious diseases and more specifically a vaccine for preventionand/or alleviation of Norovirus infections and Norovirus-relateddiseases and symptoms.

BACKGROUND

Norovirus, a single-stranded RNA virus in the Caliciviridae family, isthe primary cause of nonbacterial gastroenteritis worldwide, accountingfor 96% of all cases of viral gastroenteritis [1]. It is estimated, onaverage, that Norovirus is responsible for 19 to 21 million infectionsper year [2] and up to 200,000 deaths in children under 5 years of agein developing countries [3, 4]. Norovirus is transmitted primarilythrough the fecal-to-oral route [5] making Norovirus particularlythreatening to individuals who occupy a high density, communalenvironments such as schools, nursing homes, cruise ships, and in themilitary [6]. Norovirus is also stable ex vivo which makesdecontamination after an outbreak laborious and time consuming. Therobustness of Norovirus, along with a low infectious dose (<10 virionsper individual) [7], makes Norovirus a highly infectious virus withdramatic socio-economic impacts. This disease burden strongly indicatesthe need for an effective vaccine; however, currently there is noFDA-approved Norovirus vaccine available.

Norovirus is distributed among at least five different genogroups GI,GII, GIII, GIV, and GV. Only genogroups I, II, and IV are infectious tohumans, with GI and GII being most prevalent [8, 9]. Recently, genogroupII has become the most prevalent, accounting for 81.4% of Norovirusoutbreaks worldwide [10]. Each genogroup is subdivided further intogenoclusters. Full-length genomic sequencing of various Norovirusstrains indicate that Norovirus can vary by 3% to 31% within genogroupsand 49% to 54% between genogroups [11]. Due to this wide variation,development of a broadly effective vaccine remains a challenge as theantibodies from humans immunized against one genogroup do not crossreact with Noroviruses from other genogroups [12].

The success of virus-like particles (VLPs) as vaccine antigens has beendemonstrated by the licensure of hepatitis B virus VLP and humanpapilloma-virus VLP vaccines. Extensive research has focused on thedevelopment of Norovirus VLPs as vaccine antigens that can be deliveredparenterally, orally, or mucosally [13, 14]. Clinical evidence hasdemonstrated that Norovirus VLPs administered orally or intranasallywere well tolerated and modestly immunogenic [15, 16]. Additionalstudies have employed recombinant expression techniques to produceNorovirus VLPs using baculovirus and tobacco mosaic virus, demonstratingthat VLPs can be produced in a commercial scale with comparablestructure and immunogenicity of Norovirus VLPs produced in a traditionalway [18, 19].

Previous studies have shown that administration of Norovirus VLPsthrough the nasal cavity is able to induce systemic immunity as well asboth local and distal mucosal immunity [20, 21]. Furthermore, theincorporation of Norovirus VLPs with GELVAC nasal dry powder formulationelicits a greater immune response than antigen alone [20]. GELVAC is thedry powder formulation with GELSITE, which is an Aloe vera L.-derivedpolysaccharide polymer with mucoadhesive properties. In the presence ofdivalent cations, GELVAC is capable of in-situ gelation which improvesmucosal residence time of intranasally administered vaccines [22].

Although previous studies have shown promises of Norovirus VLPs as apotential vaccine, there is still a great need to produce a Norovirusvaccine that is multivalent, targeting the wide variation of Norovirusstrains. Moreover, the vaccine should be suitable for multiple routes ofadministrations in order to minimize the number of invasive injections.In addition, a vaccine in a form a dry power is preferred over atraditional liquid form as a dry powder can be stably stored at a roomtemperature for a long period.

SUMMARY OF INVENTION

The present invention relates to formulations of a dry powder Norovirusvaccine comprising one, two or more antigens from different genogroupsof Noroviruses. In some embodiments, the Norovirus vaccine formulationis monovalent, comprising genogroup GII VLP antigens. In someembodiment, the Norovirus vaccine formulation is multivalent, containingmultiple Norovirus VLP antigens derived from multiple genogroups ofNorovirus. In certain embodiments, the vaccine formulation is amultivalent Norovirus vaccine comprising two Norovirus VLP antigens fromGI and GII Noroviruses, respectively. In certain embodiments, thevaccine formulation is a multivalent Norovirus vaccine comprising threeNorovirus VLP antigens from GI, GII and GIV Noroviruses respectively. Insome embodiments, Norovirus VLP antigens are recombinant VLPs.Recombinant Norovirus virus-like particles are obtained by expressingthe virus-like particles in an expression system selected from a groupconsisting of viruses, baculovirus expression systems, tobacco mosaicvirus vector systems, prokaryotic cells, E. coli systems, yeast (S.cerevisiae), eukaryotic expression systems, Sf9 insect cells, mammaliancells, HEK 293 and CHO cells.

Formulations of a dry powder Norovirus vaccine may further compriseanionic polysaccharide. In some embodiments, anionic polysaccharide issodium polygalacturonate. Sodium polygalacturonate is an Aloe veraL.-derived polysaccharide polymer with mucoadhesive properties. In thepresence of divalent cations, the dry powder formulation of thiscompound improves mucosal residence time of administered vaccines. Insome embodiments, a formulation of a dry powder Norovirus vaccine isproduced as one powder formulation with a mixture of two or moreNorovirus VLP antigens. In another embodiment, the vaccine is formulatedas a combination of two or more monovalent vaccine powders with eachcontaining one Norovirus VLP antigen.

The present invention further provides methods of producing a dry powderNorovirus vaccine that is multivalent. The methods may include alyophilization-milling method. In another embodiment, the methodscomprise a spray-drying method. In some embodiments, the sodiumpolygalacturonate comprises at least 0.1% (w/w). In some embodiments,the Norovirus virus-like particle comprises about 1 μg to 100 μg of thevaccine formulation.

The present invention further provides methods of immunization againstNorovirus infections which comprises at least one immunization via aparenteral and/or a mucosal route. In some embodiments, one or moreimmunizations via a mucosal route is followed by one or moreimmunizations via a parenteral route or vice versa, to maximize bothmucosal and systemic immune responses and protection against Norovirusinfection. In some embodiments, a dry powder vaccine is used for bothparenteral and mucosal immunizations, i.e., the mucosal immunization isperformed directly with the dry powder vaccine by intranasal delivery,whereas the parenteral immunization with the reconstituted dry powdervaccine by intramuscular (IM) injection. An increase in Norovirusspecific antibodies and Norovirus neutralizing antibodies in the subjectfollowing immunization is indicative of active immunity againstNorovirus in the subject.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 : Transmission electron microscopy of Norovirus VLPs. GI (A) andGII.4 (B) VLPs were dissolved in water and imaged at 150,000×magnification (scale bar 100 μm). VLP particles were spherical inappearance at the expected size of 23 nm to 38 nm.

FIG. 2 : Thermal stability evaluation of Norovirus VLPs using SYPROOrange. VLPs were diluted in 4× SYPRO orange solution and the melt curvewas analyzed using a fluorescent thermocycler. Data is plotted as thechange in Fluorescence per unit Temperature. A. Norovirus GI VLP meltcurve. B. Norovirus GII.4 VLP melt curve.

FIG. 3 : Thermal stability evaluation of Norovirus VLPs using captureELISA. VLP samples (0.2 μg/mL) were treated at varying temperatures for5 minutes. Each sample was then analyzed by capture ELISA.

FIG. 4 : SDS-PAGE and western blot analysis of GELVAC GI (A) and GII.4(B) vaccine powders. Vaccine powders were reconstituted and analyzed bySDS-PAGE and western blot to confirm the presence of VLPs. Order fromleft to right: 100 μg, 50 μg, 15 μg, 5 μg, 1 μg, 0.1 μg, 0, referencestandard. Both GI and GII had observable bands at ˜55 kDa, consistentwith the size of VP1 capsid protein.

FIG. 5 : Serum Norovirus-specific IgG production following intranasalimmunization with GELVAC vaccine powder. Female Hartley guinea pigs wereimmunized intranasally with 20 mg of powder formulation containingvarious amounts of VLP on days 0 and 21. Serum samples were collected ondays 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B)Norovirus-specific IgG antibodies. *P<0.05 as compared to the placebocontrol group.

FIG. 6 : Serum Norovirus-specific IgG1 and IgG2 production followingintranasal immunization with GELVAC vaccine powder. Female Hartleyguinea pigs were immunized intranasally with 20 mg of powder formulationcontaining various amounts of VLP on days 0 and 21. Serum samples werecollected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) andGII.4 (B) Norovirus-specific IgG1 and IgG2 antibodies.

FIG. 7 : Serum Norovirus specific IgA production following intranasalimmunization with GELVAC vaccine powder. Female Hartley guinea pigs wereimmunized intranasally with 20 mg of powder formulation containingvarious amounts of VLP on days 0 and 21. Serum samples were collected onday 56 and analyzed for GI (A) and GII.4 (B) Norovirus-specific IgAantibodies.

FIG. 8 : Neutralizing antibody production following intranasalimmunization with GELVAC vaccine powder. Female Hartley guinea pigs wereimmunized intranasally with 20 mg of powder formulation containingvarious amounts of VLP on days 0 and 21. Serum samples were collected ondays 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B)neutralizing antibodies. *P<0.05 as compared to the placebo controlgroup.

FIG. 9 : Vaginal Norovirus-specific IgG production following intranasalimmunization with GELVAC vaccine powder. Female Hartley guinea pigs wereimmunized intranasally with 20 mg of powder formulation containingvarious amounts of VLP on days 0 and 21. Vaginal lavages samples werecollected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) andGII.4 (B) Norovirus-specific IgG antibodies. *P<0.05 as compared to theplacebo control group.

FIG. 10 : Serum and vaginal Norovirus specific IgG production after 2intranasal immunizations on day 0 and 21 followed by a parenteralimmunization on day 42 of GELVAC vaccine powder. Female Hartley guineapigs were immunized intranasally with 20 mg of powder formulationcontaining 100 μg of either GI or GII.4 VLP on days 0 and 21. On day 42,animals were immunized via an intramuscular (IM) injection of 20 mg ofpowder formulation containing 100 μg of GI or GII.4 VLP followingreconstitution with water. Vaginal lavages samples were collected ondays 0, 14, 21, 42, and 56 and analyzed for Norovirus VLP specific IgGantibodies in serum (A) and Norovirus VLP specific IgG antibodies fromvaginal swabs derived (B). *P<0.05 as compared to the placebo controlgroup.

FIG. 11 : Serum Norovirus-specific IgG and IgA production followingintranasal immunization with GELVAC monovalent and bivalent vaccinepowders. Female Hartley guinea pigs were immunized intranasally with 20mg of a bivalent vaccine powder formulation containing various amountsof GI and GII.4 VLPs on days 0 and 21. Serum samples were collected onday 0, 14, 21, 42, and 56 and analyzed for specific IgG antibodiesagainst GI (A) and GII.4 (B). Serum samples were also analyzed forspecific IgA antibodies against GI (C) and GII.4 (D). Error bars areprovided as geometric standard error. *p<0.05 as compared to the placebocontrol group.

FIG. 12 : Serum Norovirus-specific IgG1 and IgG2 production followingintranasal administration with GELVAC dry powder monovalent and bivalentvaccine. Serum samples were analyzed for Norovirus-specific IgG1antibodies against GI (A) and GII.4 (B), and Norovirus-specific IgG2antibodies against GI (C) and GII.4 (D).

FIG. 13 : Neutralizing antibody production following intranasalimmunization with GELVAC dry powder monovalent and bivalent vaccine.Female Hartley guinea pigs were immunized intranasally with 20 mg of abivalent vaccine powder formulation containing various amounts of GI andGII.4 VLPs on days 0 and 21. Serum samples were collected on days 0, 14,21, 42, and 56 and analyzed for GI (A) and GII.4 (B) neutralizingantibodies. Error bars are provided as geometric standard error. *p<0.05as compared to the placebo control group.

FIG. 14 : Mucosal Norovirus-specific antibody production followingintranasal immunization with GELVAC dry monovalent and powder bivalentvaccine. Female Hartley guinea pigs were immunized intranasally with 20mg of a bivalent vaccine powder formulation containing various amountsof GI and GII.4 VLPs on days 0 and 21. Vaginal lavage samples werecollected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) andGII.4 (B) Norovirus-specific antibodies. On day 56, animals wereeuthanized and intestinal lavage samples were analyzed for GI (C) andGII.4 (D) Norovirus-specific antibodies. Error bars are provided asgeometric standard error. *p<0.05 as compared to the placebo controlgroup.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes formulations of a Norovirus vaccine in aform of a dry powder, methods of producing such vaccine, and methods ofperforming immunization by administering such vaccine.

In order that the disclosure may be more readily understood, certainterms are first defined. These definitions should be read in light ofthe remainder of the disclosure and as understood by a person ofordinary skill in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by a person of ordinary skill in the art. Additionaldefinitions are set forth throughout the detailed description.

The articles “a” and “an,” as used herein, should be understood to mean“at least one,” unless clearly indicated to the contrary. The phrase“and/or,” when used between elements in a list, is intended to meaneither (1) that only a single listed element is present, or (2) thatmore than one element of the list is present. For example, “A, B, and/orC” indicates that the selection may be A alone; B alone; C alone; A andB; A and C; B and C; or A, B, and C. The phrase “and/or” may be usedinterchangeably with “at least one of” or “one or more of” the elementsin a list.

Norovirus

The present invention provides a formulation comprising at least twoNorovirus virus-like particle antigens. “Norovirus” herein refers tomembers of the genus Norovirus of the family Caliciviridae. In someembodiments, Norovirus includes a group of viruses that cause acutegastroenteritis in human and can be infectious to mammals including, butnot limited to, human. Norovirus may include at least five genogroups(GI-GV) defined by nucleic acid and amino acid sequences known in theart [40]. In some embodiments, Norovirus refers to a subset ofgenogroups. In certain embodiment, Norovirus refers to GI and GII.genogroups. In certain embodiments, Norovirus refers to GI, GII and GIVgenogroups.

A number of examples of Norovirus is known in the art. The examplesinclude, but not limited to, Norwalk virus, Southampton virus, DesertShield virus, and Hawaii virus. New strains of Norovirus are routinelydiscovered [41]. Use of a combination of Norovirus genogroups such as GIand GII or synthetic constructs representing combinations or portionsthereof are considered in some embodiments.

Norovirus may refer to recombinant Norovirus virus-like particles(VLPs). Norovirus VLPs are structurally similar and immunogenic asnative Norovirus, but lack the viral RNA genome of Norovirus that isrequired for infection. “Virus-like particles” or “VLPs” herein refer tovirus-like particles or fragments thereof, produced using methods knownin the art [18, 19]. In some embodiments, VLPs are produced usingbaculovirus or tobacco mosaic virus [18, 19, 23].

“Norovirus antigen” or “antigen” herein refers to any form of proteinsor peptides of Norovirus VLPs and fragments thereof, that elicit immuneresponse in vivo. Norovirus VLPs may contain Norovirus capsid proteinsor fragments thereof such as, but not limited to, VP1 and VP2. In someembodiments, Norovirus antigen comprises Norovirus VLPs. In someembodiments, Norovirus VLPs may be monovalent or multivalent. As usedherein, “monovalent” refers to antigens derived from a single genogroupof Norovirus. “Multivalent” refers to antigens derived from two or moregenogroups of Norovirus. For example, if the formulation used herein isreferred as multivalent, the formulation comprises antigens derived fromdifferent genogroups of Norovirus. When Norovirus VLPs are multivalent,Norovirus VLPs may have capsid proteins or derivatives such as VP1 andVP2 from different genogroups of Norovirus. A combination of monovalentor multivalent Norovirus VLPs may be used in a formulation of aNorovirus vaccine. In those embodiments, the resulting vaccine isreferred as multivalent, comprising Norovirus VLPs derived fromdifferent genogroups of Norovirus. A multivalent vaccine is bivalent,when it comprises two Norovirus VLPs from two different genogroups;trivalent, when it comprises three Norovirus VLP from three differentgenogroups.

Antigen Preparation

As used herein, antigens may be isolated and purified from organisms asnaturally occurred. Antigens may be produced by recombinant techniques.For example, Norovirus VLPs can be produced from cells such asprokaryotic or eukaryotic cells. Those cells include, but not limitedto, E. coli, S. cerevisiae, insect cells such as Sf9, and mammaliancells such as HEK293 cells and CHO cells. In some embodiments, anantigen is a recombinant Norovirus VLP derived from GI and/or GIIgenogroups. The recombinant Norovirus VLPs may be expressed usingbaculovirus or tobacco mosaic virus [18, 19]. In some embodiments,recombinant Norovirus VLPs are expressed and produced from plants suchas Nicotiana benthamiana, as described previously [23]. Briefly,clarified leaf extracts containing Norovirus VLPs are filtered andconcentrated. The extracts further run through a sepharose column,allowing the recovery of the VLPs. Endotoxins and other impurities maybe further removed by a fractionation. In some embodiments, the purityof Norovirus VLPs is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or100%. Norovirus VLPs as used herein preferably do not interfere with theefficacy of each VLP to elicit immunogenicity in vivo when used incombination.

Vaccine Formulation

As used herein, “vaccine” or “vaccine formulation” refers to aformulation containing Norovirus antigens that can be administered tomammals including human and elicit immune response in vivo. The vaccineformulation of this invention may prevent and/or ameliorate an infectionof Norovirus. The vaccine formulation may reduce at least one symptomrelated to Norovirus infection. The vaccine formulation may furtherenhance the efficacy of another dose of Norovirus antigen. As usedherein, “immunogenicity,” “immunogenic response,” or “immune response”refers to humoral and/or cell-mediated immune response. Humoral responseleads to production of antibodies from B lymphocytes. Cell-mediatedimmune response refers to response mediated by T lymphocytes or othercells such as macrophages.

In some embodiments, Norovirus vaccine is formulated as a form of a drypowder, capable of being administered by a mucosal route. In someembodiments, the dry powder vaccine is delivered via an intranasal wayusing an intranasal delivery device. The dry powder vaccine formulationmay be administered as a dry powder form or optionally be reconstitutedin an aqueous solution prior to administration to mammals. Thus, thevaccine formulation may be soluble in an aqueous solution. The aqueoussolution includes, but not limited to, water and saline buffer. Otherroutes to administer the vaccine formulation are considered in someembodiments, and include, but not limited to, dermal and parenteralmethods. In some embodiments, the vaccine is delivered by intramuscularinjection. Detailed routes and options to administer the formulation ofthe present invention will be discussed in immunization methods section.

The present invention includes a formulation of Norovirus vaccine thatis monovalent or multivalent. A multivalent Norovirus vaccine may beformulated as one powder formulation containing one multivalentNorovirus VLP antigen, or at least two monovalent or multivalentNorovirus VLP antigens. Alternatively, the multivalent vaccine may beformulated as a mixture of at least two dry powder formulations, eachcontaining one monovalent or multivalent Norovirus VLP antigen. Thus,Norovirus vaccine as used herein may comprise one or more Norovirus VLPantigens derived from different genogroups of Norovirus. In certainembodiments, Norovirus vaccine comprises two Norovirus VLP antigensderived from different genogroups of Norovirus. In those embodiments,Norovirus VLPs may be derived from GI and GII. Norovirus VLPs may bepresent from 0.01 μg to 1,000 μg per 20 mg of dry powder vaccineformulation, depending on the desired dose. In some embodiments,Norovirus VLPs are 10 μg to 50 μg per 20 mg of dry powder vaccineformulation.

A formulation of Norovirus vaccine as used herein may further compriseat least one or more excipients. Excipients used in the formulationpreferably do not interfere with Norovirus VLPs. In some embodiments,the excipient further enhances the therapeutic efficacy of the vaccineformulation by increasing mucosal residence time of administered vaccineformulation. The formulation of the present invention may comprises atleast one or more excipients categorized in the type of including, butnot limited to, preservatives, viscosity adjusting agents, tonicityadjusting agents, and buffering agents.

The formulation in a form of a dry powder may also contain one or moreexcipients. In some embodiments, the vaccine formulation comprises apolymer with mucoadhesive properties. In certain embodiments, thevaccine formulation comprises anionic polysaccharides. Anionicpolysaccharides include, but not limited to, dextran, guar gum, ben gum,methyl cellulose, and sodium polygalacturonate. In some embodiments, thevaccine formulation comprises sodium polygalacturonate and/or GELSITE.GELSITE is a chemically and functionally distinct high molecular weightanionic polysaccharide (sodium polygalacturonate) extracted from an Aloevera L. An exemplary method to extract a polymer from Aloe vera L. asused herein is incorporated as a reference (U.S. Pat. No. 7,705,135).Sodium polygalacturonate and GELSITE may be used herein interchangeably.The dry powder vaccine formulation may contain GELSITE in amounts of atleast 0.01%, 0.1%, 0.25%, 0.5%, or 1% (w/w). In certain embodiments, thedry powder vaccine formulation contains 0.25% (w/w) of GELSITE. “GELVACNorovirus vaccine” refers to a dry powder vaccine formulation comprisingGELSITE and at least one Norovirus VLP antigen.

Because the formulation of the present invention may comprise one ormore excipients with mucoadhesive properties, the formulation may notrequire immune adjuvants such as alum adjuvants. In some embodiments,the vaccine formulation may further comprise excipients such as, but notlimited to, povidone and lactose. Povidone (polyvinylpyrrolidone) isroutinely used in the pharmaceutical industry as a synthetic polymervehicle for dispersing and suspending drugs. Lactose is also a commonlyused excipient in the pharmaceutical industry.

Production Methods

Norovirus vaccine formulation of the present invention may be producedas a form of a dry powder and stored anhydrous until it is ready to beused. Various methods to dry a formulation are known in the art [42].The methods include, but not limited to, precipitation, crystallization,jet milling, spray-drying and lyophilizing (freeze-drying). Downstreamoperations may be further required, such as drying, milling and sieving.In some embodiments, the formulation can be freeze-dried, producingpowders with desirable characteristics. Cryo-milling may be furtherrequired in order to produce a homogenous mixture. An exemplarylyophilized-milling method is incorporated herein as a reference (U.S.Pat. No. 8,074,906). Alternatively, the formulation may be produced as apowder by a spray-drying method. Once it is in a form of a dry powder,Norovirus vaccine may have an average diameter particle size from 1 μmto 100 μm.

In some embodiments, at least two Norovirus VLP antigens derived fromdifferent genogroups of Norovirus are added in the formulation in orderto produce a multivalent dry powder Norovirus vaccine. In someembodiments, at least three Norovirus VLP antigens from differentNorovirus genogroups are added in the formulation in order to produce amultivalent dry powder vaccine. In another embodiment, a multivalent drypowder Norovirus vaccine is generated by mixing at least two drypowders, each containing one Norovirus VLP antigen. Drying constitutesdesiccating, dehydrating, or substantially dehydrating the formulation,such that a dry powder formulation is prepared. In some embodiments,each dried formulation may be milled using a mortar and pestle under acontrolled, low-humidity (<10% RH) environment, and the formulation isoptionally passed through a 70 μm filter to sterilize the formulation.

Immunization Methods

The amount of antigen in each antigenic or vaccine formulation dose isselected as an amount which induces a robust immune response withoutsignificant, adverse side effects. In general, the dose administered toa subject, in the context of the present invention should be sufficientto effect a beneficial therapeutic response in the subject over time, orto induce the production of antigen-specific antibodies. Thus, thevaccine formulation is administered to a patient in an amount sufficientto elicit an immune response to the specific antigens and/or toalleviate, reduce, or cure symptoms and/or complications from thedisease or infection. An amount adequate to accomplish this is definedas a “therapeutically effective dose.”

The vaccine formulation of the present invention may be administeredthrough such as, but not limited to, mucosal, dermal, and parenteralroutes. Exemplary detailed routes further include, but not limited to,oral, topical, subcutaneous, intranasal, intravenous, intramuscular,intranasal, sublingual, transcutaneous, subdermal, intradermal, andsuppository routes. The vaccine formulation may be administered as aform of a dry power or reconstituted in an aqueous solution prior toadministration. In some embodiments, at least two immunizations aregiven to a subject at once or separated by a few hours, days, months, oryears. In some embodiments, one or more immunizations are administeredby a mucosal route or a parenteral route. In some embodiments, one ormore immunizations via a mucosal route is followed by one or moreimmunizations via a parenteral route or vice versa, to maximize bothmucosal and systemic immune responses and protection against Norovirusinfection. In some embodiments, a dry powder vaccine is used for bothparenteral and mucosal immunizations, i.e., the mucosal immunization isperformed directly with the dry powder vaccine by intranasal delivery,whereas the parenteral immunization with the reconstituted dry powdervaccine by intramuscular injection.

As mentioned above, the vaccine formulation of the invention may beadministered to a subject to reduce the risk of Norovirus infectionprior to any future exposure to Norovirus, ameliorate and/or treatsymptoms of Norovirus infection. Symptoms of Norovirus infection arewell known in the art and include, but not limited to, nausea, vomiting,diarrhea, stomach cramping, a low-grade fever, headache, chills, muscleaches, and fatigue. The invention encompasses a method of inducing animmune response in a subject not exposed to Norovirus at the time ofadministration of the vaccine formulation of this invention.Alternatively, the formulation may be administered to a subjectcurrently experiencing a Norovirus infection such that at least onesymptom associated with Norovirus infection is alleviated and/or reducedafter administration. Successful immunization with the vaccine confersan active immunity against Norovirus in the immunized subject. Atherapeutically effective dose for immunizing a subject with the vaccineis one that results in the generation of specific antibodies against thevaccinated antigen. Additionally, a therapeutically effective dose forimmunizing a subject with the vaccine is one that results in increase inNorovirus neutralizing antibodies in the subject. Increase in specificantibodies against Norovirus antigens in the serum and mucosa of thesubject confers active immunity. Similarly, generation of Norovirusneutralizing antibody in the subject confers active immunity against aNorovirus infection. Increase in the titer of specific antibody isusually proportional to the degree of protective immunity conferred bythe vaccine. A reduction in a symptom may be determined subjectively orobjectively, e.g., self-assessment by a subject, by a clinician'sassessment or by conducting an appropriate assay or measurementincluding, but not limited to, body temperature, a level of Norovirusinfection, antibody titer, and T cell counts.

EXAMPLES

The following examples provide illustrative embodiments of thedisclosure. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the disclosure. Such modifications and variations areencompassed within the scope of the disclosure. The Examples do not inany way limit the disclosure.

Example 1 GI and GII Vaccine Formulation

Recombinant Norovirus GI and GII VLPs expressed in Nicotiana benthamianawere obtained from Kentucky Bioprocessing (Owensboro, KY) as previouslydescribed [23] and used for powder formulations. Alternatively,recombinant Norovirus GI and GII VLPs expressed and purified from Sf9insect cells using the baculovirus expression system were also used [18,19].

The GELVAC vaccine powders were made with a lyophilization-millingmethod. Liquid formulations were first prepared using a formulation thatis comprised of the recombinant VLP in a solution with GELSITE polymer,povidone and lactose. They were then lyophilized. Followinglyophilization, dried formulation contain 0.25% (w/w) GELSITE, 99%lactose and 0.05% povidone and 0 μg to 100 μg (based on ELISA data) ofVLP per 20 mg of formulation, depending on the desired dose. The GI andGII VLPs were added together to the formulation to produce themultivalent powder or individually to produce the monovalent powders.The multivalent powder can also be produced by mixing together two ormore monovalent powders. Each dried formulation could be milled using amortar and pestle under a controlled, low-humidity (<10% RH) environmentand passed through a 70 μm filter. The powder formulations can be madeusing a spray-drying apparatus as well. Powders were stored in sealedcontainers under desiccation at room temperature until use.

Example 2 Characterization of Vaccine Formulations VLP Characterization

GI and GII VLP stocks were analyzed for the presence of intact VLPs bytransmission electron microscopy prior to powder manufacturing. Theresults confirmed the presence of intact VLPs of the expected sizes (38nm) for both GI and GII VLP stocks (FIG. 1 ).

VLP stability was established by determining the melt temperature ofNorovirus VLPs with SYPRO Orange. Briefly, SYPRO Orange (Sigma-Aldrich,St. Louis, MO) was diluted in PBS to make a final 4× concentration ofSYPRO Orange. Each VLP was diluted in 4× SYPRO Orange to a finalconcentration of 1 mg/mL. Each sample was then placed in a fluorescentthermocycler and was run through a 25° C.-95° C. gradient while readingthe fluorescent signal. The derivative of the signal was determined bytaking the difference between successive points in the fluorescentsignal. The noise was reduced by using a 4-point moving average filter.Melt curve plots are shown for GI (FIG. 2A) and GII (FIG. 2B). GI VLPshad two melt peaks, one minor peak at 43° C. and a major peak at 65° C.GII VLPs showed a major peak at 65° C. The major peak observed in theGII VLP melt curve is consistent with the major peak for GI VLPs. Theseresults demonstrated that the VLP antigens were stable at temperaturesup to 65° C.

VLP stability was also evaluated based on antigenicity. Norovirus VLPswere incubated at various temperatures and tested in a capture ELISA.Mouse monoclonal IgG2 anti-Norovirus antibodies (Maine Biotech, MAB228(GI); MAB227 (GII)) diluted 1:2000 in PBS were coated on Nunc MaxiSorp96-well plates (Fisher Scientific, Pittsburgh, PA) overnight at 4° C.The wells were washed 5 times with wash buffer, and then blocked for 1hr at room temperature in blocking buffer. Norovirus VLPs were dilutedin blocking buffer, and allowed to incubate on the plate at roomtemperature for 1 hr. The wells were washed 3 times with wash buffer,followed by incubation with corresponding mouse monoclonal IgG1anti-Norovirus antibodies (Millipore, MAB80143 (GI); Maine BiotechMAB226 (GII)) diluted 1:2000 in blocking buffer for 1 hr at roomtemperature. The wells were washed 3 times with wash buffer, followed byincubation with a polyclonal anti-mouse IgG1:HRP (Abcam, Cambridge, MA)diluted 1:2000 in blocking buffer for 1 hr at room temperature. Finally,the wells were washed 3 times with wash buffer and were developed using1-step Ultra TMB according to manufacturer's protocol (ThermoScientific, Waltham, MA). The OD at 450 nm was measured and plottedagainst known VLP concentrations.

For each VLP, the OD decreased significantly at 65° C., consistent withthe major melt peaks observed via SYPRO Orange (FIG. 3 ). Thus, thedenaturing of the VLP antigens at high temperatures was correlated withthe loss of antigenicity. These results also confirm the specificity ofthe capture ELISA to intact VLPs, which was used to determine theantigen dose content of each GELVAC vaccine powder for use in the animalstudies.

GELVAC Vaccine Powder Characterization

The GELVAC vaccine powder was manufactured through a manual millingprocess under nitrogen gas. Laser diffraction particle size distributionconfirmed the volumetric mean particle size to be 24 μm to 37 μm for allpowders, which was determined using a laser diffraction particle sizeanalyzer with a liquid module (Beckman Coulter L513 320, Pasadena, CA).Furthermore, the d10 for the powders was approximately 5 μm for allpowders, thus minimizing the amount of powders (<5 μm) which can reachdeep lung. A representative particle distribution result for eachantigen can be found in Table 1. The mean particle diameter for the GIVLP formulation was 29.73 μm and for GII VLP formulation was 25.2 μm.

TABLE 1 Representative volumetric particle size distribution of GI andGII monovalent vaccine powders Vaccine powders Mean d10 d50 d90 GI 29.73μm 5.08 μm 25.23 μm 59.25 μm GII 25.20 μm 5.49 μm 22.97 μm 49.32 μm

To determine the presence of Norovirus VP1 for each GI and GII in thevaccine powders, SDS-PAGE and western blotting was performed (FIG. 4 ).Both SDS-PAGE and western blotting show the presence of a major band at˜55 kDa in all powders. These results are consistent with the expectedsize of VP1. Due to the lack sensitivity, VLPs were not visible in doses<15 μg in either SDS-PAGE or western blot. The multiple bands presenthave been observed previously and confirmed to be due to possibletruncation of the VP1 protein [26, 27]. These results also show relativeamounts of VP1 were consistent with total VLP concentration.

Capture ELISAs were used to quantify the VLP dose content of eachvaccine powder. GI and GII capture ELISAs were performed with a 15 μgdose formulated vaccine. 10 mg of each 15 μg VLP dose powders weredissolved in 1 mL of water. Each powder was tested in the capture ELISAand compared to the VLP reference standard. Using a 4-PL fit, theantigen concentration of each powder was then quantified based on theweight of vaccine powder (Table 2). The capture ELISA was establishedusing the GI- and GII-specific monoclonal antibodies. Nocross-reactivity was observed between these two genogroups. In addition,no interference was observed with the powder formulation excipients.

TABLE 2 Testing of GELVAC GI and GII vaccine powders for antigen contentExpected Antigen Dose Observed Antigen Dose Vaccine Powders (μg VLP/mgPowder) (μg VLP/mg Powder) GI VLP 15 μg/10 mg 15.46 μg/10 mg GII VLP 15μg/10 mg 16.16 μg/10 mg

Example 3 Immunogenicity of Vaccine Formulations Immunogenicity ofGELVAC GI and GII Powders

The immunogenicity of a GELVAC vaccine powder formulated with GI VLP hasbeen reported previously [20]. To further these studies, antigendose-dependent immune responses were investigated with GELVAC vaccinepowders with GI or GII VLPs in female (250 g) Hartley guinea pigs.Animals were dosed with varying amounts of Norovirus GI or GII VLPs(Table 3) or with a multivalent GI/GII VLP vaccine (50 μg each of GI andGII VLP, twice on days 0 and 21.

TABLE 3 Monovalent Vaccine Animal Experimental Design Monovalent GuineaPig Studies Total Antigen per Group # n Vaccination (μg)* 1 4 100 2 4 503 4 15 4 4 5 5 4 1 6 4 0.1 7 4 0 *Animals were immunized with a total of20 mg of powder via both nares. Each nare received 10 mg of powder orhalf of the total antigen dose.

The vaccine powders were administered intranasally using Aptar Unit DoseSpray (UDS) Devices (Aptar Pharma, Congers, NY), one per nare with halfof the total antigen dose per nare (10 mg total powder per nare). Thecontrol group was administered the same amount of a placebo powderformulation. Serum and vaginal lavage samples were collected from theanimals on days 0 (preimmunization), 21, 42 and 56.

Clinical Observations

There were no abnormal clinical findings in the guinea pigs aftervaccination. There were also no statistical differences in body weightbetween the control and test vaccine groups (data not shown). Throughoutthe 56 day duration of the immunogenicity study, all vaccines were welltolerated by the test subjects. One animal in the 100 μg dose group forGI antigen was lost after the second immunization on day 21. Uponautopsy examination, death was attributed to anesthesia and/or the bloodcollection procedure.

Serum Antibody Response

Serum samples were analyzed for Norovirus VLP specific IgG by ELISA.Norovirus GI or GII VLPs (2 μg/mL) in PBS were incubated on NuncMaxiSorp 96-well plates (Fisher Scientific) for 4 hrs at roomtemperature. The plates were blocked overnight at 4° C. in blockingbuffer. All samples were diluted in blocking buffer and serially diluted2-fold down the plate. Samples were allowed to incubate at roomtemperature for 1 hr. The wells were washed 5 times with wash buffer,followed by incubation with anti-guinea pig IgG-HRP secondary antibodies(Southern Biotech, Birmingham, AL) at 1:1000 for 1 hr at roomtemperature. The wells were washed 5 times with wash buffer. The wellswere developed using 1-step Ultra TMB according to manufacturer'sprotocol. End-point titers were reported as the reciprocal of thehighest dilution that produced an OD of 0.1 above background. A positivecontrol serum generated in guinea pigs against GI or GII VLP wasincluded in each test run to confirm reproducibility.

The total antigen specific IgG antibodies present in the serum exhibiteda dose-dependent increase with both GI and GII vaccine powders (FIG. 5). Compared to the control group, serum IgG titers increased on day 21and peaked by day 42 at all doses greater than 1 μg. By day 42, GI IgGtiters increased by >600-fold for all dose groups of ≥15 μg and GII IgGtiters increased by >300-fold for all dose groups of ≥5 μg. A dose ofthe vaccine which corresponds to >100-fold increase in antigen-specificIgG titers in the subject confers active immunity against a Norovirusinfection. An increase in antigen-specific antibody titer between 10- to1000-fold is indicative of active immunity against the virus. There wereno significant differences between 15 μg and 100 μg doses for GI andbetween 5 μg and 100 μg doses for GII. The lowest dose that elicited anantigen specific IgG response was 1 μg for both GI and GII whichcorresponded to a titer of 495 and 320 on day 56, respectively. Asexpected, all doses above 0.1 μg exhibited a boosting effect after thesecond dosing on day 21 with both GI and GII powders. These resultsshowed that the VLPs formulation with GELVAC nasal powder were highlyimmunogenic and significant antibody production can be induced withGELVAC nasal powders with GI VLP at 15 μg and GII VLP at 5 μg.

Overall, both GI and GII vaccine powders induced a dose dependentantibody response. Serum antigen specific IgG antibody production wascorrelated with amounts of both GI and GII VLP antigens present in thepowders and reached a maximal level at 15 μg to 50 μg of VLP antigen.Administration of higher doses of VLPs did not result in significantlyhigher levels of antigen specific IgGs. It is important to note that theboosting effect on systemic and mucosal IgGs was observed for each VLPantigen after the second dose on day 21.

Both IgG1 and IgG2 subclasses were also analyzed using the serum samplesfrom each group (FIG. 6 ). Both IgG1 and IgG2 exhibited similar responseprofiles as the total IgG described above. The IgG2 titers wereapparently higher than IgG1 titers with both GI and GII VLP powders,especially at low dose levels (1 and 5 μg) with a difference of up to100 fold (FIG. 6 ).

Antigen-specific IgA serum levels were also investigated. At day 56,anti-GI and anti-GII VLP IgA antibodies were observed at all doses thatwere administered when compared to mock dose controls, except for the1.0 μg dose group with GII (FIG. 7 ). They also showed an overall trendof higher levels at higher antigen doses. These results showed that theVLP formulations with GELVAC nasal powder were highly immunogenic andsignificant antigen specific antibody can be induced with GELVAC nasalpowders with GI at 15 μg and GII VLP at 5 μg.

Serum Neutralization Antibody Response

Previous studies have demonstrated that Norovirus VLP-specificantibodies can block the binding of Norovirus VLP to ABH histo-bloodgroup antigen (HBGA) in a strain specific manner [28]. HBGAs arecarbohydrates ubiquitously expressed on mucosal tissues and red bloodcells that have been implicated as natural receptors for Norovirusbinding and entry, demonstrating that blockade of HBGA interactions withVLPs may prevent Norovirus infection [29]. To this end, antigen specificantibodies were investigated for their ability to inhibit the binding ofthe Norovirus VLPs to porcine gastric mucin. The neutralizing antibodiespresent in the serum exhibited a dose-dependent response similar to thatobserved for antigen specific IgG antibody titers (FIG. 8 ). Compared tothe control, GI neutralizing antibody titers were elevated in 15 μg dosegroup by day 21, in 15 μg, 50 μg, and 100 μg dose groups by day 42, andin all dose groups greater than 1 μg by day 56. GII neutralizingantibody titers were elevated for the 100 μg dose group by day 21,elevated by day 42 in the 5 μg, 50 μg, and 100 μg dose groups, andelevated in all dose groups greater than 1 μg by day 56. By day 42, GIneutralizing antibody titers increased by >5-fold for all dose groupsof >5 μg and GII neutralizing antibody titers increased by >10-fold forall dose groups >1 μg, consistent with the findings with serum IgGtiters. There were no significant differences between 5 μg and 100 μgdoses for both GI and GII at day 56. The lowest dose that produced adetectable neutralization titer at day 56 was 5 μg for both GI and GII.The highest neutralizing antibody titers at day 56 occurred in the 15 μgdose group for GI and 100 μg dose group for GII. As expected, all groupsabove 5 μg for both GI and GII exhibited a boosting effect after thesecond dose on day 21. These results showed that the neutralizingantibody titers followed a similar dose-dependent response to thatobserved for the total serum IgG titers.

Production of antigen specific serum antibodies is necessary but notsufficient for protection against Norovirus infection. However,production of serum antibodies that neutralize the HBGA binding siteshas been largely accepted as a surrogate marker for efficacy andcorrelates well with protection in humans and chimpanzees [17, 37, 38].The present disclosure demonstrates that the GELVAC vaccine powdercontaining either GI or GII VLPs administered intranasally was capableof producing antibodies in guinea pigs that inhibited the binding of theVLPs to pig gastric mucin. The serum levels of these neutralizingantibodies were correlated with the amount of GI or GII VLP administeredto the guinea pigs. In a similar fashion that was observed for serum IgGantibodies, a boosting effect in the neutralizing antibody titers afterthe second dose was also observed. However, a larger amount of VLPantigen was required for the production of neutralizing antibodies ascompared to the induction of total specific IgG antibodies. A dose of 15μg for both GI and GII VLP antigen was required to maximize theproduction of neutralizing antibodies. These results correlated wellwith the serum IgG titers and further support a maximally efficaciousdose of 15 μg for each genogroup. A dose of the vaccine whichcorresponds to at least >100-fold increase in antigen-specific IgGtiters and >5-fold increase in neutralizing antibody titers isconsidered effective to confer active immunity against a Norovirusinfection.

Mucosal Antibody Response

To investigate the mucosal immune response at various antigen doses,mucosal antibody titers were evaluated in the reproductive tracts withvaginal lavage (FIG. 9 ). GI vaginal antibody titers were elevated in 50μg dose group by day 21 and in all dose groups greater than 1 μg by day56. GII vaginal antibody titers were elevated in the 5 μg, 50 μg, and100 μg dose groups by day 42. The lowest dose that elicited a mucosalIgG response was 5 μg for both GI and GII. The highest vaginal antibodytiters occurred at 15 μg for GI and 100 μg for GII. These results showedthat vaginal IgG antibody titers exhibited a dose-dependent responsethat reached a significantly higher level at 15 μg and 50 μg for GI andGII, respectively.

Antigen specific IgG antibody production in the vaginal tract showedsimilar trends to what was observed for both antigen specific IgGantibodies and neutralizing antibodies detected in serum. Presence ofmucosal IgG antibodies is most likely conferred through transudation ofserum IgG antibodies [39]. These results demonstrate that the GELVACvaccine powder is capable to inducing a mucosal response along with aneutralizing antibody response.

Example 4 Immunizations Via Both Mucosal and Parenteral Routes

Animals received the first two doses of GELVAC Norovirus VLP powdervaccine containing GI or GII. 4 VLP intranasally as described above ondays 0 and 21. For the third dose, the same amount of the vaccinepowders were reconstituted with water and administered by intramuscular(IM) injection on day 42. The serum and vaginal antibodies were measuredas described above. As shown in FIG. 10 , the total antigen specific IgGantibodies present in the serum and vaginal lavage increased with themonovalent GI and GII vaccine powders. The monovalent powder vaccinesused in this experiment were manufactured with either GI or GIINorovirus VLPs. Compared to the control group, serum IgG titersincreased on day 21 and were higher on day 42. By day 42, serum IgGtiters increased by >200-fold for both GI and GII antigens.

Serum VLP specific titers further increased by an additional 10-foldafter the IM immunization for both GI and GII (FIG. 10 ). VLP specificIgG titers were also further increased in the vaginal lavage samples. Atday 42, following two IN immunizations, 2- to 4-fold increases inantigen specific IgG titers were observed in the vaginal lavage for bothGI and GII VLPs. Following the IM immunization, VLP specific titers invaginal lavage increased by 10-fold for both GI and GII VLPs. Thus, arelatively larger increase in the mucosal antibodies was obtained afterthe IM immunization. These results showed that the Norovirus VLPsformulation with GELVAC nasal powder were highly immunogenic and capableof inducing significant systemic and mucosal antibody productionfollowing mucosal or intranasal immunization, and importantly, bothsystemic and mucosal antibody responses can be further increased by anadditional parenteral or IM immunization

The present disclosure further shows that immune responses induced by aNorovirus vaccine can be further enhanced by immunization via bothparenteral and mucosal routes. As shown herein, animals were firstimmunized with the Norovirus VLP powder vaccine intranasally twicefollowed by an additional immunization via IM injection with thereconstituted powder vaccine, and a significant increase in bothsystemic and mucosal immune responses was obtained after the additionalimmunization by IM injection. Thus, a Norovirus vaccine can beadministered with one or more doses via a mucosal route followed by oneor more doses via a parenteral route or vice versa to further enhancethe immune responses. There may be an interval of 2 to 4 weeks betweenthe two routes of immunization. It is preferred that the first dose isadministered by the mucosal route and the second dose by the parenteralroute. The dry powder vaccine is particularly advantageous forimmunization by the combinatorial routes, i.e., the mucosal immunizationcan be performed directly with the dry powder vaccine by intranasaldelivery, whereas the parenteral immunization by IM injection isperformed with the same dry powder vaccine after reconstitution, whichmay simply be carried out with sterile water.

Example 5 Immunizations With Bivalent Vaccines Immunogenicity of GelVacGI and GII Powders

The immunogenicity of the GELVAC vaccine powder formulated with GI andGII VLPs, individually, has been reported previously [14, 16]. Tofurther these studies, dose-dependent immune responses were investigatedwith bivalent GELVAC vaccine formulations containing GI and GIINorovirus VLPs. Animals were dosed with varying amounts of the bivalentNorovirus GI and GII VLPs (Table 4) on days 0 and 21. Serum and vaginallavage samples were collected from the animals on days 0(preimmunization), 21, 42 and 56. Intestinal lavage was collected afterthe termination of the study (day 56).

TABLE 4 Bivalent Vaccine Animal Experimental Design Antigen Sample rVLPPresentation Collection Group Dose Schedule Schedule Size GroupFormulation (μg) * (Study Day) (Study Day) (n) 1 Control 0 0, 21 0, 7,14, 21, 42, 56 4 2 GI Antigen 50 0, 21 0, 7, 14, 21, 42, 56 4 3 GIIAntigen 50 0, 21 0, 7, 14, 21, 42, 56 4 4 GI & GII 5 0, 21 0, 7, 14, 21,42, 56 4 Antigen 5 GI & GII 15 0, 21 0, 7, 14, 21, 42, 56 4 Antigen 6 GI& GII 50 0, 21 0, 7, 14, 21, 42, 56 4 Antigen 7 GI & GII 100 0, 21 0, 7,14, 21, 42, 56 4 Antigen * Animals were immunized with a total of 20 mgof powder via both nares. Each nare received 10 mg of powder or half ofthe total antigen dose.

Serum Antibody Response

Serum samples were analyzed for Norovirus VLP specific IgG by ELISA. Thetotal antigen specific IgG antibodies present in the serum exhibited adose-dependent increase with both GI and GII vaccine powders (FIG. 11 ).Compared to the control group, serum IgG titers increased on day 14 anda further increase was observed on days 21 and 42 at all doses >5 μg. Byday 14, GI IgG titers increased by at >4-fold compared to controls.Further increases in GI IgG titers were observed at day 21 compared today 14. Similar results were observed for GII IgG titers at day 14 and21. At day 42 GI IgG titers increased by >600-fold compared to day 21for all dose groups of ≥5 μg and GII IgG titers increased by >300-foldcompared to day 21 for all dose groups of ≥5 μg. There were nosignificant differences between 15 μg and 100 μg doses for GI andbetween 5 μg and 100 μg doses for GII. The lowest dose that elicited anantigen specific IgG response was 5 μg for both GI and GII whichcorresponded to a titer of 30800 and 245840 on day 56, respectively.Antigen specific IgA serum levels were also investigated. At day 56,anti-GI and anti-GII VLP IgA antibodies were observed at all doses thatwere administered when compared to the mock dose controls (FIG. 11 ).They also exhibited an overall trend of higher levels at higher antigendoses.

The IgG1 and IgG2 subclasses were also analyzed using the pooled serumsamples from each group (FIG. 12 ). As shown, GI and GII IgG2 specificantibody titers were observed at day 14 but not IgG1 specific titers. GIand GII IgG2 specific titers were also shown to be higher than GI or GIIIgG1 specific titers at day 21. IgG1 and IgG2 Boosting effects were alsoobserved for both GI and GII at day 42. Overall, the IgG2 titers werehigher than IgG1 titers for both GI and GII VLPs (FIG. 12 ). Theseresults show that the bivalent GI/GII VLP vaccine formulations werehighly immunogenic and capable of producing a wide range of antibodyresponses.

Serum Neutralization Antibody Response

Antigen specific antibodies were investigated for their ability toinhibit the binding of the Norovirus VLPs to porcine gastric mucin. Theneutralizing antibodies present in the serum exhibited a dose-dependentresponse similar to that observed for antigen specific IgG antibodytiters (FIG. 13 ). Compared to the control, GI neutralizing antibodytiters were elevated in the 15 μg, 50 μg, and 100 μg dose groups by day42 with similar titers observed at day 56. GII neutralizing antibodytiters were elevated for all groups by day 42 with similar titersobserved at day 56. By day 42, GI neutralizing antibody titers increasedby >50-fold for all dose groups ≥24 μg and GII neutralizing antibodytiters increased by >190-fold for all dose groups ≥5 μg, consistent withthe findings with serum IgG titers. There were no significantdifferences between 5 μg and 100 μg doses for both GI and GII at day 56.The lowest dose that produced a detectable neutralization titer at day56 was 15 μg for GI and 5 μg for GII. These results showed that theneutralizing antibody titers followed a similar dose-dependent responseto that observed for the total serum IgG titers.

Mucosal Antibody Response

To investigate the mucosal immune response at various antigen doses,mucosal antibody titers were evaluated in the reproductive tracts andintestines (FIG. 14 ). GI vaginal antibody titers were elevated in 50 μgand 100 μg dose group by day 21 and in all dose groups greater than 5 μgby day 56. GII vaginal antibody titers were elevated in the 5 μg, 15 μg,50 μg, and 100 μg dose groups by day 42. The lowest dose that elicited amucosal IgG response was 5 μg for both GI and GII. The highest vaginalantibody titers occurred at 50 μg for both GI and GII. These resultsshowed that vaginal IgG antibody titers exhibited a dose-dependentresponse. GI and GII specific IgG titers were also observed in theintestines at day 56 (FIG. 14 C and D). As shown, antibody titers wereobserved in all treatment groups for both GI and GII specificantibodies.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method for producing a multivalent dry powder Norovirusvaccine composition suitable for both parenteral and mucosal routes ofadministration comprising: a. obtaining a first solution comprising aNorovirus virus-like particle (VLP) antigen of genogroup GI; b.introducing to the first solution, an anionic polysaccharide; c. dryingthe first solution with the anionic polysaccharide to a substantiallynon-aqueous state, thereby producing a first dry powder formulation; d.obtaining a second solution comprising a Norovirus VLP antigen ofgenogroup GII; e. introducing to the second solution an anionicpolysaccharide; f. drying the second solution with the anionicpolysaccharide to a substantially non-aqueous state, thereby producing asecond dry powder formulation; g. combining the first and second drypowder formulations to form a multivalent dry powder Norovirus vaccineformulation.
 2. The method of claim 1, wherein the drying is bylyophilization.
 3. The method of claim 1, wherein the drying is by spraydrying.
 4. The method of claim 1, wherein the Norovirus VLP of genogroupGI and Norovirus VLP of genogroup GII are obtained by expressing therecombinant virus-like particles in an expression system selected fromthe group consisting of: prokaryote cells, eukaryote cells, E. colicells, S. cerevisiae cells, insect cells, mammalian cells, HEK293 cells,CHO cells, tobacco mosaic virus, and baculovirus.